Randomized libraries of zinc finger proteins

ABSTRACT

The present invention relates to methods of using libraries of randomized zinc finger proteins to identify genes associated with selected phenotypes.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/337,216 filed Jan. 6, 2003, which is a continuation of U.S. patentapplication Ser. No. 09/731,558 filed Dec. 6, 2000, now U.S. Pat. No.6,503,717, which is a continuation-in-part of U.S. patent applicationSer. No. 09/456,100 filed Dec. 6, 1999, now abandoned; the disclosuresof which are herein incorporated by reference in their entireties.

This application is related to U.S. Ser. No. 09/229,007, filed Jan. 12,1999, and U.S. Ser. No. 09/229,037, filed Jan. 12, 1999, and U.S. Ser.No. 09/395,448, filed Sep. 14, 1999, herein each incorporated byreference in their entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not applicable.

FIELD OF THE INVENTION

The present invention relates to methods of using libraries ofrandomized zinc finger proteins to identify genes associated withselected phenotypes.

BACKGROUND OF THE INVENTION

A. Using Libraries to Identify Genes Associated with a SelectedPhenotype

Identification of gene function is a critical step in the selection ofnew molecular targets for drug discovery, gene therapy, clinicaldiagnostics, agrochemical discovery, engineering of transgenic plants,e.g., with novel resistance traits or enhanced nutritionalcharacteristics, and genetic engineering of prokaryotes and higherorganisms for the production of industrial chemicals, biochemicals, andchemical intermediates. Historically, library screening methods havebeen used to screen large numbers of uncharacterized genes to identify agene or genes associated with a particular phenotype, e.g.,hybridization screening of nucleic acid libraries, antibody screening ofexpression libraries, and phenotypic screening of libraries.

For example, molecular markers that co-segregate with a disease trait ina segment of patients can be used as nucleic acid probes to identify, ina library, the gene associated with the disease. In another method,differential gene expression in cells and nucleic acid subtraction canbe used to identify and clone genes associated with a phenotype in thetest cells, where the control cells do not display the phenotype.However, these methods are laborious because the screening step reliesheavily on conventional nucleic acid cloning and sequencing techniques.Development of high throughput screening assays using these methodswould therefore be cumbersome.

An example of phenotypic screening of libraries is discovery oftransforming oncogenes (see, e.g., Goldfarb et al., Nature 296:404(1982)). Oncogenic transformation can be observed in NIH 3T3 cells byassaying for loss of contact inhibition and foci formation. cDNAexpression libraries from transformed cells are introduced intountransformed cells, and the cells were examined for foci formation. Thegene associated with transformation is isolated by clonal propagationand rescue of the expression vector. Unfortunately, this method islimited by phenotype and can only be used to assay for transdominantgenes.

Advances in the field of high throughput screening have increased thecell types and phenotypes that can be investigated using libraryscreening methods. Viral vectors such as retroviral, adenoviral, andadenoviral associated vectors have been developed for efficient nucleicacid delivery to cells (see, e.g., U.S. Pat. No. 5,173,414; Tratschin etal., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell.Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, Proc. Nat'l Acad. Sci.USA 81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828(1989); Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al.,J. Virol. 66:1635-1640 (1992); Sommerfelt et al., Virol. 176:58-59(1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J.Virol. 65:2220-2224 (1991); and PCT/US94/05700). Cells can bephenotypically analyzed either one at a time, using flow cytometry, orin arrayed clonal populations, using liquid handling robots. Thesetechniques allow a sufficient number of library members to be tested fora wide range of potential phenotypes.

Currently, libraries of random molecules are being used with phenotypicscreening for the discovery of genes associated with a particularphenotype. For example, random peptide or protein expression librariesare being used to block specific protein-protein interactions andproduce a particular phenotype (see, e.g., Caponigro et al., Proc. Nat'lAcad. Sci USA 95:7508-7513 (1998); WO 97/27213; and WO 97 27212). Inanother method, random antisense nucleic acids or ribozymes are used toinactivate a gene and produce a desired phenotype (see, e.g., WO99/41371 and Hannon et al., Science 283:1125-1126 (1999)).

The main shortcoming of these methods is the inherent inefficiency ofthe random molecules, which vastly increases the size of the library tobe screened. Even with a known target nucleic acid or protein, literallyhundreds of antisense, ribozyme, or peptide molecules must beempirically tested before identifying one that will inhibit geneexpression or protein-protein interactions. Since the random librarymust be enormous to produce sufficient numbers of active molecules, hugenumbers of cells must be screened for phenotypic changes. For unknowngene and protein targets, the rarity of effective, bioactive peptides,antisense molecules, or ribozyme molecules imposes significantconstraints on high throughput screening assays. Furthermore, thesemethods can be used only for inhibition of gene expression, but not foractivation of gene expression. This feature limits identification ofgene function to phenotypes present only in the absence of geneexpression.

Therefore, efficient high throughput library screening methods allowingrandom inhibition or activation of uncharacterized genes would be ofgreat utility to the scientific community. These methods would findwidespread use in academic laboratories, pharmaceutical companies,genomics companies, agricultural companies, chemical companies, and inthe biotechnology industry.

B. Zinc Finger Proteins as Transcriptional Regulators

Zinc finger proteins (“ZFPs”) are proteins that bind to DNA in asequence-specific manner and are typically involved in transcriptionregulation. Zinc finger proteins are widespread in eukaryotic cells. Anexemplary motif characterizing one class of these proteins (the Cys₂His₂class) is -Cys-(X)₂₋₄-Cys-(X)₁₂-His-(X)₃₋₅-His (SEQ ID NO:1) (where X isany amino acid). A single finger domain is about 30 amino acids inlength and several structural studies have demonstrated that it containsan alpha helix containing the two invariant histidine residuesco-ordinated through zinc with the two cysteines of a single beta turn.To date, over 10,000 zinc finger sequences have been identified inseveral thousand known or putative transcription factors. Zinc fingerproteins are involved not only in DNA-recognition, but also in RNAbinding and protein-protein binding. Current estimates are that thisclass of molecules will constitute the products of about 2% of all humangenes.

The X-ray crystal structure of Zif268, a three-finger domain from amurine transcription factor, has been solved in complex with its cognateDNA-sequence and shows that each finger can be superimposed on the nextby a periodic rotation and translation of the finger along the main DNAaxis. The structure suggests that each finger interacts independentlywith DNA over 3 base-pair intervals, with side-chains at positions −1,2, 3 and 6 on each recognition helix making contacts with respective DNAtriplet sub-site.

The structure of the Zif268-DNA complex also suggested that the DNAsequence specificity of a zinc finger protein could be altered by makingamino acid substitutions at the four helix positions (−1, 2, 3 and 6) ona zinc finger recognition helix, using, e.g., phage display experiments(see, e.g., Rebar et al., Science 263:671-673 (1994); Jamieson et al.,Biochemistry 33:5689-5695 (1994); Choo et al., Proc. Natl. Acad. Sci.U.S.A. 91:11163-11167 (1994); Greisman & Pabo, Science 275:657-661(1997)). For example, combinatorial libraries were constructed with zincfinger proteins randomized in either the first or middle finger. Therandomized zinc finger proteins were then isolated with altered targetsites in which the appropriate DNA sub-site was replaced by an alteredDNA triplet. Correlation between the nature of introduced mutations andthe resulting alteration in binding specificity gave rise to a set ofsubstitution rules for rational design of zinc finger proteins withaltered binding specificity. These experiments thus demonstrated thatrandomized zinc finger proteins could be made, which demonstratedaltered target sequence specificity.

Recombinant zinc finger proteins, often combined with a heterologoustranscriptional activator or repressor domain, have also shown efficienttranscriptional regulation of transiently expressed reporter genes incultured cells (see, e.g., Pomerantz et al., Science 267:93-96 (1995);Liu et al., Proc. Natl. Acad. Sci. U.S.A. 94:5525-5530 1997); and Beerliet al., Proc. Natl. Acad. Sci. U.S.A. 95:14628-14633 (1998)). Forexample, Pomerantz et al., Science 267:93-96 (1995) designed a novel DNAbinding protein by fusing two fingers from Zif268 with a homeodomainfrom Oct-1. The hybrid protein was then fused with either atranscriptional activator or repressor domain for expression as achimeric protein. The chimeric protein was reported to bind a targetsite representing a hybrid of the subsites of its two components. Thechimeric DNA binding protein also activated or repressed expression of areporter luciferase gene having a target site.

Liu et al., Proc. Natl. Acad. Sci. U.S.A. 94:5525-5530 (1997)constructed a composite zinc finger protein by using a peptide spacer tolink two component zinc finger proteins, each having three fingers. Thecomposite protein was then further linked to transcriptional activationor repression domains. The resulting chimeric protein bound to a targetsite formed from the target segments bound by the two component zincfinger proteins. The chimeric zinc finger protein activated or repressedtranscription of a reporter gene having the target site.

Beerli et al., Proc. Natl. Acad. Sci. U.S.A. 95:14628-14633 (1998)constructed a chimeric six finger zinc finger protein fused to either aKRAB, ERD, or SID transcriptional repressor domain, or the VP16 or VP64transcriptional activation domain. This chimeric zinc finger protein wasdesigned to recognize an 18 bp target site in the 5′ untranslated regionof the human erbB-2 gene. This construct both activated and repressed atransiently expressed reporter luciferase construct linked to the erbB-2promoter.

In addition, a recombinant zinc finger protein was reported to repressexpression of an integrated plasmid construct encoding a bcr-ab1oncogene (Choo et al., Nature 372:642-645 (1994)). Phage display wasused to select a variant zinc finger protein that bound to the selectedtarget segment. The variant zinc finger protein thus isolated was thenreported to repress expression of a stably transfected bcr-ab1 constructin a cell line. To date, these zinc finger protein methods have focusedon regulation of either single, transiently expressed, known genes, oron regulation of single, known exogenous genes that have been integratedinto the genome.

SUMMARY OF THE INVENTION

The present application therefore provides for the first time methods ofusing libraries of randomized zinc finger proteins to screen largenumbers of genes, for identifying a gene or genes associated with aselected phenotype. These libraries of randomized zinc finger DNAbinding proteins have the ability to regulate gene expression with highefficiency and specificity. Because zinc finger proteins provide areliable, efficient means for regulating gene expression, the librariesof the invention typically have no more than about 10⁶ to about 10⁷members. This manageable library size means that libraries of randomizedzinc finger proteins can be efficiently used in high throughputapplications to quickly and reliably identify genes of interest that areassociated with any given phenotype.

In one aspect, the present invention provides a method of identifying agene or genes associated with a selected phenotype, the methodcomprising the steps of: (a) providing a nucleic acid library comprisingnucleotide sequences that encode partially randomized zinc fingerproteins; (b) transducing cells with expression vectors, each comprisinga nucleotide sequence from the library; (c) culturing the cells so thatzinc finger proteins are expressed in the cells, wherein the zinc fingerproteins modulate gene expression in at least some of the cells; (d)assaying the cells for a selected phenotype and determining whether ornot the cells exhibit the selected phenotype; and (e) identifying, incells that exhibit the selected phenotype, the gene or genes whoseexpression is modulated by expression of a zinc finger protein, whereinthe gene so identified is associated with the selected phenotype.

In one embodiment, the zinc finger protein has three, four, or fivefingers. In another embodiment, the library is made by finger grafting,DNA shuffling, or codon doping. In another embodiment, the librarycomprises no more than about 10⁶ clones, no more than about 10⁷ clones,or no more than about 10⁸ clones.

In one embodiment, the cells are physically separated, individual poolsof cells and each individual pool of cells is transduced with anexpression vector comprising a nucleotide sequence from the library. Inanother embodiment, the physical separation of the pools of cells isaccomplished by placing each pool of cells in a separate well of a 96,384, or 1536 well plate. In another embodiment, the cells are assayedfor the selected phenotype using liquid handling robots. In anotherembodiment, the cells are pooled together and transduced in a batch. Inanother embodiment, the cells are assayed for the selected phenotypeusing flow cytometry. In one embodiment, the cells are selected from thegroup consisting of animal cells, plant cells, bacterial cells,protozoal cells, mammalian cells, human cells, or fungal cells.

In one embodiment, zinc finger proteins are fusion proteins comprisingone or two regulatory domains, e.g., a transcriptional repressor, amethyl transferase, a transcriptional activator, a histoneacetyltransferase, and a histone deacetylase. In another embodiment, theregulatory domain is VP16 or KRAB. In another embodiment, the zincfinger proteins comprise a Zif268 backbone.

In one embodiment, modulation of gene expression is repression of geneexpression. In another embodiment, modulation of gene expression isactivation of gene expression. In one embodiment, expression of the zincfinger proteins is controlled by administration of a small molecule,e.g., tetracycline.

In one embodiment, the expression vectors are a viral vector, e.g., aretroviral expression vector, a lentiviral expression vector, anadenoviral expression vector, or an AAV expression vector.

In one embodiment, the selected phenotype is related to cancer,nephritis, prostate hypertrophy, hematopoiesis, osteoporosis, obesity,cardiovascular disease, or diabetes. In one embodiment, genes that aresuspected of being associated with the selected phenotype are identifiedby comparing differential gene expression patterns in the presence andabsence of expression of the zinc finger protein. In another embodiment,differential gene expression patterns are compared using anoligonucleotide array. In another embodiment genes that are suspected ofbeing associated with the selected phenotype are identified by usingzinc finger proteins from the library of randomized zinc finger proteinsto probe YAC or BAC clones. In another embodiment, genes that aresuspected of being associated with the selected phenotype are identifiedby scanning genomic sequences for target sequences recognized by zincfinger proteins from the library of randomized zinc finger proteins. Inanother embodiment, genes that are suspected of being associated withthe selected phenotype are identified by cross-linking the zinc fingerprotein to DNA with which it is associated, followed byimmunoprecipitation of the zinc finger protein and sequencing of theDNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a zinc finger protein gene assembly using PCR.

FIG. 2 shows a diagram of making random zinc finger proteins with DNAshuffling.

FIG. 3 shows the life cycle of an adeno-associated virus.

FIG. 4 shows high throughput, arrayed generation of AAV-ZFP vectorlibraries.

FIG. 5 shows assaying for a phenotype of interest.

DETAILED DESCRIPTION OF THE INVENTION

As described herein, the present invention provides libraries ofrandomized zinc finger proteins used in screening assays to identify agene or genes associated with a selected phenotype. These libraries ofrandomized zinc finger proteins can be readily used to either up- ordown-regulate gene expression. No target DNA sequence information isrequired to create a random DNA binding domain. This feature makes thezinc finger protein technology ideal for screening for genes that areassociated with a desired phenotype. One can simply create a library ofrandomized zinc finger-based DNA binding domains, create chimeric up anddown-regulating transcription factors and test the effect of up ordown-regulation on the phenotype under study (transformation, responseto a cytokine etc.) by switching the genes on or off in any modelsystem.

Additionally, greater experimental control can be imparted by zincfinger proteins than can be achieved by more conventional methods suchas antisense, ribozyme, and peptide applications. This control isavailable because the expression and/or function of an engineered zincfinger protein can be placed under small molecule control. Examples ofthis approach are provided, e.g., by the Tet-On system, theecdysone-regulated system, and the RU-486 system (see, e.g., Gossen &Bujard, Proc. Natl. Acad. Sci. U.S.A. 89:5547 (1992); Oligino et al.,Gene Ther. 5:491-496 (1998); Wang et al., Gene Ther. 4:432-441 (1997);Neering et al., Blood 88:1147-1155 (1996); and Rendahl et al., Nat.Biotechnol. 16:757-761 (1998)).

In the present invention, a nucleic acid library of about no more than10⁶ to 10⁷ partially randomized zinc finger proteins is made, usingtechniques such as codon doping, gene shuffling, and finger grafting.Often, a three-fingered zinc protein is used in the methods of theinvention. Cells are then transfected with the library for expression ofa zinc finger protein clone. Preferably, the zinc finger proteins areintroduced into the cell using viral expression vectors, e.g.,retroviral or adenoviral-based vectors. The cells are then assayed forchanges in the phenotype of choice. Cells can be assayed one by one,using techniques such as flow cytometry, or in pools of arrayed clonalpopulations, using liquid handling robots (see Example section, below).

Examples of assay systems for changes in phenotype include, e.g.,transformation assays, e.g., changes in proliferation, anchoragedependence, growth factor dependence, foci formation, and growth in softagar; apoptosis assays, e.g., DNA laddering and cell death, expressionof genes involved in apoptosis; signal transduction assays, e.g.,changes in intracellular calcium, cAMP, cGMP, IP3, changes in hormoneand neurotransmitter release; receptor assays, e.g., estrogen receptorand cell growth; growth factor assays, e.g., EPO, hypoxia anderythrocyte colony forming units assays; enzyme production assays, e.g.,FAD-2 induced oil desaturation; pathogen resistance assays, e.g.,insect, bacterial, and viral resistance assays; chemical productionassays, e.g., penicillin production; transcription assays, e.g.,reporter gene assays; and protein production assays, e.g., VEGF ELISAs.

Those cells exhibiting an altered phenotype are selected for furtherstudy, in which the genes associated with the change in phenotype areidentified and isolated. The genes are identified and isolated, e.g.,using differential gene expression analysis with microarrays; reversegenetics; e.g., identification of genes using zinc finger proteins toprobe YAC or BAC clones and using zinc finger proteins to scan genomicsequences; subtractive hybridization; differential cDNA cloningfrequencies, subtractive hybridization; by cloning ESTs from cells ofinterest; by identifying genes that are lethal upon knockout; byidentifying genes that are up- or down-regulated in response to aparticular developmental or cellular event or stimuli; by identifyinggenes that are up- or down-regulated in certain disease and pathogenicstates; by identifying mutations and RFLPs; by identifying genesassociated with regions of chromosomes known to be involved in inheriteddiseases; by identifying genes that are temporally regulated, e.g., in apathogenic organism; differences based on SNPs, etc.

In one embodiment, the zinc finger protein is linked to at least one ormore regulatory domains, described in detail below. Preferred regulatorydomains include transcription factor repressor or activator domains suchas KRAB and VP16, co-repressor and co-activator domains, DNAmethyltransferases, histone acetyltransferases, histone deacetylases,and endonucleases such as Fok1. For repression of gene expression, oftensimple steric hindrance of transcription initiation is sufficient.

Such assays for candidate genes allow for discovery of novel human andveterinary therapeutic and diagnostic applications, including thediscovery of novel drugs, for, e.g., treatment of genetic diseases,cancer, fungal, protozoal, bacterial, and viral infection, ischemia,vascular disease, arthritis, immunological disorders, etc. In addition,the methods of the invention can be used in the agricultural industryfor the identification of commercially relevant plant genes, and can beused to engineer bacteria and other organisms to produce industrialchemicals and pharmaceuticals.

Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

“Partially randomized” zinc finger proteins refers to a zinc fingerprotein where at least some of the amino acids of any individual fingerare generated randomly and are not preselected (e.g., the four criticalamino acids of finger 1), or wherein at least one finger or part of afinger from a known zinc finger protein is randomly combined withanother heterologous finger or part of a finger from a known zinc fingerprotein. Typically, a standard zinc finger protein backbone from amammalian zinc finger protein such as SP1 or Zif268 is used to make thepartially random protein, with the fingers either partially or fullyrandomized via random codon selection. In some cases the codons arepartially randomized, e.g., to eliminate termination codons (see Table2, below). Partially random zinc finger proteins include fullyrandomized zinc finger proteins. In one embodiment, amino acids −1, 2,3, and 6 of a finger are randomly selected.

A “gene associated with a selected phenotype” refers to a cellular,viral, bacterial, protozoal, fungal, animal, plant, episomal,chloroplastic, or mitochondrial gene, where modulation of geneexpression using a randomized zinc finger protein causes a change in theselected phenotype. This term also refers to a microbial or viral genethat is part of a naturally occurring microbial or viral genome in amicrobially or virally infected cell. The microbial or viral genome canbe extrachromosomal or integrated into the host chromosome. This termalso encompasses endogenous and exogenous genes, as well as cellulargenes that are identified as expressed sequence tags (“ESTs”). An assayof choice is used to identify genes associated with a selected phenotypeupon regulation of gene expression with a zinc finger protein. The genesare typically identified via methods such as gene expressionmicroarrays, differential cDNA cloning frequencies, subtractivehybridization and differential display methods. The genes associatedwith a selected phenotype are then subjected to target validation usingengineered zinc finger proteins (see, e.g., copending patent applicationU.S. Ser. No. 09/395,448, filed Sep. 14, 1999).

A “selected phenotype” refers to any phenotype, e.g., any observablecharacteristic such as a physical, chemical, or functional effect thatcan be measured in an assay such as changes in cell growth,proliferation, morphology, enzyme function, signal transduction,expression patterns, downstream expression patterns, reporter geneactivation, hormone release, growth factor release, neurotransmitterrelease, ligand binding, apoptosis, and product formation. Such assaysinclude, e.g., transformation assays, e.g., changes in proliferation,anchorage dependence, growth factor dependence, foci formation, andgrowth in soft agar; apoptosis assays, e.g., DNA laddering and celldeath, expression of genes involved in apoptosis; signal transductionassays, e.g., changes in intracellular calcium, cAMP, cGMP, IP3, changesin hormone and neurotransmitter release; receptor assays, e.g., estrogenreceptor and cell growth; growth factor assays, e.g., EPO, hypoxia anderythrocyte colony forming units assays; enzyme production assays, e.g.,FAD-2 induced oil desaturation; pathogen resistance assays, e.g.,insect, bacterial, and viral resistance assays; chemical productionassays, e.g., penicillin production; transcription assays, e.g.,reporter gene assays; and protein production assays, e.g., VEGF ELISAs.

The term “zinc finger protein” or “ZFP” refers to a protein having DNAbinding domains that are stabilized by zinc. The individual DNA bindingdomains are typically referred to as “fingers” A zinc finger protein hasat least one finger, typically two fingers, three fingers, four fingers,five fingers, or six fingers or more. Each finger binds from two to fourbase pairs of DNA, typically three or four base pairs of DNA. A zincfinger protein binds to a nucleic acid sequence called a target site ortarget segment. Each finger typically comprises an approximately 30amino acid, zinc-coordinating, DNA-binding subdomain. An exemplary motifcharacterizing one class of these proteins (Cys₂His₂ class) is-Cys-(X)₂₋₄-Cys-(X)₁₂-His-(X)₃₋₅-His (SEQ ID NO:1) (where X is any aminoacid). Studies have demonstrated that a single zinc finger of this classconsists of an alpha helix containing the two invariant histidineresidues co-ordinated with zinc along with the two cysteine residues ofa single beta turn (see, e.g., Berg & Shi, Science 271:1081-1085(1996)).

A “target site” is the nucleic acid sequence recognized by a zinc fingerprotein. A single target site typically has about four to about ten ormore base pairs. Typically, a two-fingered zinc finger proteinrecognizes a four to seven base pair target site, a three-fingered zincfinger protein recognizes a six to ten base pair target site, a sixfingered zinc finger protein recognizes two adjacent nine to ten basepair target sites, and so on for proteins with more than six fingers.The target site is in any position that allows regulation of geneexpression, e.g., adjacent to, up- or downstream of the transcriptioninitiation site; proximal to an enhancer or other transcriptionalregulation element such as a repressor (e.g., SP-1 binding sites,hypoxia response elements, nuclear receptor recognition elements, p53binding sites, etc.), RNA polymerase pause sites; and intron/exonboundaries. The term “adjacent target sites” refers to non-overlappingtarget sites that are separated by zero to about 5 base pairs.

“K_(d)” refers to the dissociation constant for the compound, i.e., theconcentration of a compound (e.g., a zinc finger protein) that giveshalf maximal binding of the compound to its target (i.e., half of thecompound molecules are bound to the target) under given conditions(i.e., when [target]<<K_(d)), as measured using a given assay system(see, e.g., U.S. Pat. No. 5,789,538). The assay system used to measurethe K_(d) should be chosen so that it gives the most accurate measure ofthe actual K_(d) of the zinc finger protein. Any assay system can beused, as long is it gives an accurate measurement of the actual K_(d) ofthe zinc finger protein. In one embodiment, the K_(d) for the zincfinger proteins of the invention is measured using an electrophoreticmobility shift assay (“EMSA”), as described herein. Unless an adjustmentis made for zinc finger protein purity or activity, the K_(d)calculations made using the methods described herein may result in anunderestimate of the true K_(d) of a given zinc finger protein.Optionally, the K_(d) of a zinc finger protein used to modulatetranscription of a candidate gene is less than about 100 nM, or lessthan about 75 nM, or less than about 50 nM, or less than about 25 nM.

“Administering” an expression vector, nucleic acid, zinc finger protein,or a delivery vehicle to a cell comprises transducing, transfecting,electroporating, translocating, fusing, phagocytosing, or ballisticmethods, etc., i.e., any means by which a protein or nucleic acid can betransported across a cell membrane and preferably into the nucleus of acell, including administration of naked DNA.

A “delivery vehicle” refers to a compound, e.g., a liposome, toxin, or amembrane translocation polypeptide, which is used to administer a zincfinger protein. Delivery vehicles can also be used to administer nucleicacids encoding zinc finger proteins, e.g., a lipid:nucleic acid complex,an expression vector, a virus, and the like.

The terms “modulating expression” “inhibiting expression” and“activating expression” of a gene refer to the ability of a zinc fingerprotein to activate or inhibit transcription of a gene. Activationincludes prevention of transcriptional inhibition (i.e., prevention ofrepression of gene expression) and inhibition includes prevention oftranscriptional activation (i.e., prevention of gene activation).

“Activation of gene expression that prevents repression of geneexpression” refers to the ability of a zinc finger protein to block theaction of or prevent binding of a repressor molecule.

“Inhibition of gene expression that prevents gene activation” refers tothe ability of a zinc finger protein to block the action of or preventbinding of an activator molecule.

Modulation can be assayed by determining any parameter that isindirectly or directly affected by the expression of the target gene.Such parameters include, e.g., changes in RNA or protein levels, changesin protein activity, changes in product levels, changes in downstreamgene expression, changes in reporter gene transcription (luciferase,CAT, β-galactosidase, β-glucuronidase, GFP (see, e.g., Mistili &Spector, Nature Biotechnology 15:961-964 (1997)); changes in signaltransduction, phosphorylation and dephosphorylation, receptor-ligandinteractions, second messenger concentrations (e.g., cGMP, cAMP, IP3,and Ca²⁺), and cell growth, etc., as described herein. These assays canbe in vitro, in vivo, and ex vivo. Such functional effects can bemeasured by any means known to those skilled in the art, e.g.,measurement of RNA or protein levels, measurement of RNA stability,identification of downstream or reporter gene expression, e.g., viachemiluminescence, fluorescence, fluorescent activated cell sorting(“FACS”), calorimetric reactions, antibody binding, inducible markers,ligand binding assays; changes in intracellular second messengers suchas cGMP and inositol triphosphate (IP3); changes in intracellularcalcium levels; cytokine release, and the like, as described herein.

To determine the level of gene expression modulation effected by a zincfinger protein, cells contacted with zinc finger proteins are comparedto control cells, e.g., without the zinc finger protein or with anon-specific zinc finger protein, to examine the extent of inhibition oractivation. Control samples are assigned a relative gene expressionactivity value of 100%. Modulation/inhibition of gene expression isachieved when the gene expression activity value relative to the controlis about 80%, preferably 50% (i.e., 0.5× the activity of the control),more preferably 25%, more preferably 5-0%. Modulation/activation of geneexpression is achieved when the gene expression activity value relativeto the control is 110%, more preferably 150% (i.e., 1.5× the activity ofthe control), more preferably 200-500%, more preferably 1000-2000% ormore.

A “transcriptional activator” and a “transcriptional repressor” refer toproteins or effector domains of proteins that have the ability tomodulate transcription, as described above. Such proteins include, e.g.,transcription factors and co-factors (e.g., KRAB, MAD, ERD, SID, nuclearfactor kappa B subunit p65, early growth response factor 1, and nuclearhormone receptors, VP16, VP64), endonucleases, integrases, recombinases,methyltransferases, histone acetyltransferases, histone deacetylasesetc. Activators and repressors include co-activators and co-repressors(see, e.g., Utley et al., Nature 394:498-502 (1998)).

A “regulatory domain” refers to a protein or a protein domain that hastranscriptional modulation activity when tethered to a DNA bindingdomain, i.e., a zinc finger protein. Typically, a regulatory domain iscovalently or non-covalently linked to a zinc finger protein to effecttranscription modulation. Alternatively, a zinc finger protein can actalone, without a regulatory domain, to effect transcription modulation.

The term “heterologous” is a relative term, which when used withreference to portions of a nucleic acid indicates that the nucleic acidcomprises two or more subsequences that are not found in the samerelationship to each other in nature. For instance, a nucleic acid thatis recombinantly produced typically has two or more sequences fromunrelated genes synthetically arranged to make a new functional nucleicacid, e.g., a promoter from one source and a coding region from anothersource. The two nucleic acids are thus heterologous to each other inthis context. When added to a cell, the recombinant nucleic acids wouldalso be heterologous to the endogenous genes of the cell. Thus, in achromosome, a heterologous nucleic acid would include an non-native(non-naturally occurring) nucleic acid that has integrated into thechromosome, or a non-native (non-naturally occurring) extrachromosomalnucleic acid.

Similarly, a heterologous protein indicates that the protein comprisestwo or more subsequences that are not found in the same relationship toeach other in nature (e.g., a “fusion protein,” where the twosubsequences are encoded by a single nucleic acid sequence). See, e.g.,Ausubel, supra, for an introduction to recombinant techniques.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (naturally occurring) form of the cell orexpress a second copy of a native gene that is otherwise normally orabnormally expressed, under expressed or not expressed at all.

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription. As used herein, a promoter typically includesnecessary nucleic acid sequences near the start site of transcription,such as, in the case of certain RNA polymerase II type promoters, a TATAelement, enhancer, CCAAT box, SP-1 site, etc. As used herein, a promoteralso optionally includes distal enhancer or repressor elements, whichcan be located as much as several thousand base pairs from the startsite of transcription. The promoters often have an element that isresponsive to transactivation by a DNA-binding moiety such as apolypeptide, e.g., a nuclear receptor, Gal4, the lac repressor and thelike.

A “constitutive” promoter is a promoter that is active under mostenvironmental and developmental conditions. An “inducible” promoter is apromoter that is active under certain environmental or developmentalconditions.

The term “operably linked” refers to a functional linkage between anucleic acid expression control sequence (such as a promoter, or arrayof transcription factor binding sites) and a second nucleic acidsequence, wherein the expression control sequence directs transcriptionof the nucleic acid corresponding to the second sequence.

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell, and optionally, integration or replication of the expressionvector in a host cell. The expression vector can be part of a plasmid,virus, or nucleic acid fragment, of viral or non-viral origin.Typically, the expression vector includes an “expression cassette,”which comprises a nucleic acid to be transcribed operably linked to apromoter. The term expression vector also encompasses naked DNA operablylinked to a promoter.

By “host cell” is meant a cell that contains a zinc finger protein or anexpression vector or nucleic acid encoding a zinc finger protein. Thehost cell typically supports the replication and/or expression of theexpression vector. Host cells may be prokaryotic cells such as E. coli,or eukaryotic cells such as yeast, fungal, protozoal, higher plant,insect, or amphibian cells, or mammalian cells such as CHO, HeLa, 293,COS-1, and the like, e.g., cultured cells (in vitro), explants andprimary cultures (in vitro and ex vivo), and cells in vivo.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms also apply to amino acid polymers in which one or more amino acidresidues is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

-   1) Alanine (A), Glycine (G);-   2) Aspartic acid (D), Glutamic acid (E);-   3) Asparagine (N), Glutamine (Q);-   4) Arginine (R), Lysine (K);-   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);-   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);-   7) Serine (S), Threonine (T); and-   8) Cysteine (C), Methionine (M)-   (see, e.g., Creighton, Proteins (1984)).    Making Libraries of Randomized Zinc Finger Proteins

Libraries of nucleic acids encoding randomized zinc finger proteins aregenerated for use in the methods of the invention. Typically, a backbonefrom any suitable Cys₂His₂ zinc finger protein, such as SP-1, SP-1C, orZIF268, is used as the scaffold for the randomized zinc finger protein(see, e.g., Jacobs, EMBO J. 11:4507 (1992); Desjarlais & Berg, Proc.Nat'l Acad. Sci. USA 90:2256-2260 (1993)). A number of methods can thenbe used to generate libraries of nucleic acids encoding the randomizedzinc finger proteins.

At least three different strategies can be used to make the random zincfinger protein libraries. The first method, called the finger orrecognition helix grafting strategy, will typically have the leastnon-functional zinc finger proteins, and the recombination is limitedonly to the existing fingers. The second method, called the codon dopingstrategy, provides the most complete randomization scheme. The thirdmethod, called the gene shuffling strategy, will generate new variantsfor all fingers. In this method, however, the mutagenesis is notcomplete but is derived from only a limited number of parental zincfinger proteins. The three randomization schemes can be used herein tobuild the randomized zinc finger protein libraries and to test thelibraries for DNA binding in vitro by using a phage display system (seeExample section, below).

In one embodiment, the method used for zinc finger protein libraryconstruction is fingertip, or recognition helix grafting. Imagine acollection of 3-bp binding zinc finger protein helices that could begrafted together in any combination. Each unique multi-fingercombination would recognize a different unique DNA sequence. The numberof different fingers used and the number of fingers attached togethercan be varied in this method. In one embodiment, the number of differentfingertips is about 10-14, optionally 12, and the number of fingers is3-5, optionally 5, and a randomized zinc finger library size to screenpreferably consists of 250,000+members.

There are about 140,000 genes in the human genome and the human genomehas about 3×10⁹ basepairs. In one embodiment, a library of five fingerzinc finger proteins made with 20 different fingertips would recognizeabout 3,200,000 different 15 basepair sequences (20⁵); a library madewith 15 different fingertips would recognize about 759,375 differentsequences; a library made with 13 fingertips would recognize about371,293 different sequences; and a library made with 12 fingertips wouldrecognize about 248,832. Using specific helices for all 64 tripletswould be sufficient to recognize any and all 15 basepair sequences.

Considering both strands of the target genome, a 15 basepair sequence isexpected to occur 0.6 times ((2.8×10⁸/4¹⁵)×2). In other words, of arandom 5-finger library, at least 60 percent of the component zincfinger proteins are expected to affect the expression of a single gene.Considering the entire genome, a random 5-finger zinc finger protein isexpected to have on average only 6 perfect binding sites.

On average, no more than one gene should be directly affected at a timeby a component zinc finger protein, and only a handful of genomicbinding sites need to be considered. In fact, the active zinc fingerprotein itself can be used to identify candidate genes either bysequence scanning or as probes to identify candidate genomic clones(i.e., from YAC or BAC clones).

In addition, any other suitable method known in the art can be used toconstruct nucleic acids encoding random zinc finger proteins, e.g.,phage display, random mutagenesis, combinatorial libraries, affinityselection, PCR, cloning from cDNA or genomic libraries, syntheticconstruction and the like. (see, e.g., U.S. Pat. No. 5,786,538; Wu etal., Proc. Nat'l Acad. Sci. USA 92:344-348 (1995); Jamieson et al.,Biochemistry 33:5689-5695 (1994); Rebar & Pabo, Science 263:671-673(1994); Choo & Klug, Proc. Nat'l Acad. Sci. USA 91:11163-11167 (1994);Choo & Klug, Proc. Nat'l Acad. Sci. USA 91: 11168-11172 (1994);Desjarlais & Berg, Proc. Nat'l Acad. Sci. USA 90:2256-2260 (1993);Desjarlais & Berg, Proc. Nat'l Acad. Sci. USA 89:7345-7349 (1992);Pomerantz et al., Science 267:93-96 (1995); Pomerantz et al., Proc.Nat'l Acad. Sci. USA 92:9752-9756 (1995); and Liu et al., Proc. Nat'lAcad. Sci. USA 94:5525-5530 (1997); Greisman & Pabo, Science 275:657-661(1997); Desj'arlais & Berg, Proc. Nat'l Acad. Sci. USA 91:11-99-11103(1994)).

Regulatory Domains

The zinc finger proteins of the invention can optionally be associatedwith regulatory domains for modulation of gene expression. The zincfinger protein can be covalently or non-covalently associated with oneor more regulatory domains, alternatively two or more regulatorydomains, with the two or more domains being two copies of the samedomain, or two different domains. The regulatory domains can becovalently linked to the zinc finger protein, e.g., via an amino acidlinker, as part of a fusion protein. The zinc finger proteins can alsobe associated with a regulatory domain via a non-covalent dimerizationdomain, e.g., a leucine zipper, a STAT protein N terminal domain, or anFK506 binding protein (see, e.g., O'Shea, Science 254: 539 (1991),Barahmand-Pour et al., Curr. Top. Microbiol. Immunol. 211:121-128(1996); Klemm et al., Annu. Rev. Immunol. 16:569-592 (1998); Klemm etal., Annu. Rev. Immunol. 16:569-592 (1998); Ho et al., Nature382:822-826 (1996); and Pomeranz et al., Biochem. 37:965 (1998)). Theregulatory domain can be associated with the zinc finger protein at anysuitable position, including the C- or N-terminus of the zinc fingerprotein.

Common regulatory domains for addition to the zinc finger proteininclude, e.g., effector domains from transcription factors (activators,repressors, co-activators, co-repressors), silencers, nuclear hormonereceptors, oncogene transcription factors (e.g., myc, jun, fos, myb,max, mad, rel, ets, bcl, mos family members etc.); DNA repair enzymesand their associated factors and modifiers; DNA rearrangement enzymesand their associated factors and modifiers; chromatin associatedproteins and their modifiers (e.g., kinases, acetylases anddeacetylases); and DNA modifying enzymes (e.g., methyltransferases,topoisomerases, helicases, ligases, kinases, phosphatases, polymerases,endonucleases) and their associated factors and modifiers.

Transcription factor polypeptides from which one can obtain a regulatorydomain include those that are involved in regulated and basaltranscription. Such polypeptides include transcription factors, theireffector domains, coactivators, silencers, nuclear hormone receptors(see, e.g., Goodrich et al., Cell 84:825-30 (1996) for a review ofproteins and nucleic acid elements involved in transcription;transcription factors in general are reviewed in Barnes & Adcock, Clin.Exp. Allergy 25 Suppl. 2:46-9 (1995) and Roeder, Methods Enzymol.273:165-71 (1996)). Databases dedicated to transcription factors areknown (see, e.g., Science 269:630 (1995)). Nuclear hormone receptortranscription factors are described in, for example, Rosen et al., J.Med. Chem. 38:4855-74 (1995). The C/EBP family of transcription factorsare reviewed in Wedel et al., Immunobiology 193:171-85 (1995).Coactivators and co-repressors that mediate transcription regulation bynuclear hormone receptors are reviewed in, for example, Meier, Eur. J.Endocrinol. 134(2):158-9 (1996); Kaiser et al., Trends Biochem. Sci.21:342-5 (1996); and Utley et al., Nature 394:498-502 (1998)). GATAtranscription factors, which are involved in regulation ofhematopoiesis, are described in, for example, Simon, Nat. Genet. 11:9-11(1995); Weiss et al., Exp. Hematol. 23:99-107. TATA box binding protein(TBP) and its associated TAF polypeptides (which include TAF30, TAF55,TAF80, TAF110, TAF150, and TAF250) are described in Goodrich & Tjian,Curr. Opin. Cell Biol. 6:403-9 (1994) and Hurley, Curr. Opin. Struct.Biol. 6:69-75 (1996). The STAT family of transcription factors arereviewed in, for example, Barahmand-Pour et al., Curr. Top. Microbiol.Immunol. 211:121-8 (1996). Transcription factors involved in disease arereviewed in Aso et al., J. Clin. Invest. 97:1561-9 (1996).

In one embodiment, the KRAB repression domain from the human KOX-1protein is used as a transcriptional repressor (Thiesen et al., NewBiologist 2:363-374 (1990); Margolin et al., Proc. Nat'l Acad. Sci. USA91:4509-4513 (1994); Pengue et al., Nucl. Acids Res. 22:2908-2914(1994); Witzgall et al., Proc. Nat'l Acad. Sci. USA 91:4514-4518(1994)). In another embodiment, KAP-1, a KRAB co-repressor, is used withKRAB (Friedman et al., Genes Dev. 10:2067-2078 (1996)). Alternatively,KAP-1 can be used alone with a zinc finger protein. Other preferredtranscription factors and transcription factor domains that act astranscriptional repressors include MAD (see, e.g., Sommer et al., J.Biol. Chem. 273:6632-6642 (1998); Gupta et al., Oncogene 16:1149-1159(1998); Queva et al., Oncogene 16:967-977 (1998); Larsson et al.,Oncogene 15:737-748 (1997); Laherty et al., Cell 89:349-356 (1997); andCultraro et al., Mol Cell. Biol. 17:2353-2359 (19977)); FKHR (forkheadin rhapdosarcoma gene; Ginsberg et al., Cancer Res. 15:3542-3546 (1998);Epstein et al., Mol. Cell. Biol. 18:4118-4130 (1998)); EGR-1 (earlygrowth response gene product-1; Yan et al., Proc. Nat'l Acad. Sci. USA95:8298-8303 (1998); and Liu et al., Cancer Gene Ther. 5:3-28 (1998));the ets2 repressor factor repressor domain (ERD; Sgouras et al., EMBO J.14:4781-4793 (1995)); and the MAD smSIN3 interaction domain (SID; Ayeret al., Mol. Cell. Biol. 16:5772-5781 (1996)).

In one embodiment, the HSV VP16 activation domain is used as atranscriptional activator (see, e.g., Hagmann et al., J. Virol.71:5952-5962 (1997)). Other preferred transcription factors that couldsupply activation domains include the VP64 activation domain (Seipel etal., EMBO J. 11:4961-4968 (1996)); nuclear hormone receptors (see, e.g.,Torchia et al., Curr. Opin. Cell. Biol. 10:373-383 (1998)); the p65subunit of nuclear factor kappa B (Bitko & Barik, J. Virol. 72:5610-5618(1998) and Doyle & Hunt, Neuroreport 8:2937-2942 (1997)); and EGR-1(early growth response gene product-1; Yan et al., Proc. Nat'l Acad.Sci. USA 95:8298-8303 (1998); and Liu et al., Cancer Gene Ther. 5:3-28(1998)).

Kinases, phosphatases, and other proteins that modify polypeptidesinvolved in gene regulation are also useful as regulatory domains forzinc finger proteins. Such modifiers are often involved in switching onor off transcription mediated by, for example, hormones. Kinasesinvolved in transcription regulation are reviewed in Davis, Mol. Reprod.Dev. 42:459-67 (1995), Jackson et al., Adv. Second MessengerPhosphoprotein Res. 28:279-86 (1993), and Boulikas, Crit. Rev. Eukaryot.Gene Expr. 5:1-77 (1995), while phosphatases are reviewed in, forexample, Schonthal & Semin, Cancer Biol. 6:239-48 (1995). Nucleartyrosine kinases are described in Wang, Trends Biochem. Sci. 19:373-6(1994).

As described, useful domains can also be obtained from the gene productsof oncogenes (e.g., myc, jun, fos, myb, max, mad, rel, ets, bcl, mosfamily members) and their associated factors and modifiers. Oncogenesare described in, for example, Cooper, Oncogenes, The Jones and BartlettSeries in Biology (2^(nd) ed., 1995). The ets transcription factors arereviewed in Waslylk et al., Eur. J. Biochem. 211:7-18 (1993) andCrepieux et al., Crit. Rev. Oncog. 5:615-38 (1994). Myc oncogenes arereviewed in, for example, Ryan et al., Biochem. J. 314:713-21 (1996).The jun and fos transcription factors are described in, for example, TheFos and Jun Families of Transcription Factors (Angel & Herrlich, eds.1994). The max oncogene is reviewed in Hurlin et al., Cold Spring Harb.Symp. Quant. Biol. 59:109-16. The myb gene family is reviewed inKanei-Ishii et al., Curr. Top. Microbiol. Immunol. 211:89-98 (1996). Themos family is reviewed in Yew et al., Curr. Opin. Genet. Dev. 3:19-25(1993).

Zinc finger proteins can include regulatory domains obtained from DNArepair enzymes and their associated factors and modifiers. DNA repairsystems are reviewed in, for example, Vos, Curr. Opin. Cell Biol.4:385-95 (1992); Sancar, Ann. Rev. Genet. 29:69-105 (1995); Lehmann,Genet. Eng. 17:1-19 (1995); and Wood, Ann. Rev. Biochem. 65:135-67(1996). DNA rearrangement enzymes and their associated factors andmodifiers can also be used as regulatory domains (see, e.g., Gangloff etal., Experientia 50:261-9 (1994); Sadowski, FASEB J. 7:760-7 (1993)).

Similarly, regulatory domains can be derived from DNA modifying enzymes(e.g., DNA methyltransferases, topoisomerases, helicases, ligases,kinases, phosphatases, polymerases) and their associated factors andmodifiers. Helicases are reviewed in Matson et al., Bioessays, 16:13-22(1994), and methyltransferases are described in Cheng, Curr. Opin.Struct. Biol. 5:4-10 (1995). Chromatin associated proteins and theirmodifiers (e.g., kinases, acetylases and deacetylases), such as histonedeacetylase (Wolffe, Science 272:371-2 (1996)) are also useful asdomains for addition to the zinc finger protein of choice. In onepreferred embodiment, the regulatory domain is a DNA methyl transferasethat acts as a transcriptional repressor (see, e.g., Van den Wyngaert etal., FEBS Lett. 426:283-289 (1998); Flynn et al., J. Mol. Biol.279:101-116 (1998); Okano et al., Nucleic Acids Res. 26:2536-2540(1998); and Zardo & Caiafa, J. Biol. Chem. 273:16517-16520 (1998)). Inanother preferred embodiment, endonucleases such as Fok1 are used astranscriptional repressors, which act via gene cleavage (see, e.g.,WO95/09233; and PCT/US94/01201).

Factors that control chromatin and DNA structure, movement andlocalization and their associated factors and modifiers; factors derivedfrom microbes (e.g., prokaryotes, eukaryotes and virus) and factors thatassociate with or modify them can also be used to obtain chimericproteins. In one embodiment, recombinases and integrases are used asregulatory domains. In one embodiment, histone acetyltransferase is usedas a transcriptional activator (see, e.g., Jin & Scotto, Mol. Cell.Biol. 18:4377-4384 (1998); Wolffe, Science 272:371-372 (1996); Tauntonet al., Science 272:408-411 (1996); and Hassig et al., Proc. Nat'l Acad.Sci. USA 95:3519-3524 (1998)). In another embodiment, histonedeacetylase is used as a transcriptional repressor (see, e.g., Jin &Scotto, Mol. Cell. Biol. 18:4377-4384 (1998); Syntichaki & Thireos, J.Biol. Chem. 273:24414-24419 (1998); Sakaguchi et al., Genes Dev.12:2831-2841 (1998); and Martinez et al., J. Biol. Chem. 273:23781-23785(1998)).

Linker domains between polypeptide domains, e.g., between two zincfinger proteins or between a zinc finger protein and a regulatorydomain, can be included. Such linkers are typically polypeptidesequences, such as poly gly sequences of between about 5 and 200 aminoacids. Preferred linkers are typically flexible amino acid subsequenceswhich are synthesized as part of a recombinant fusion protein. Forexample, in one embodiment, the linker DGGGS (SEQ ID NO:2) is used tolink two zinc finger proteins. In another embodiment, the flexiblelinker linking two zinc finger proteins is an amino acid subsequencecomprising the sequence TGEKP (SEQ ID NO:3) (see, e.g., Liu et al.,Proc. Nat'l Acad. Sci. USA 5525-5530 (1997)). In another embodiment, thelinker LRQKDGERP (SEQ ID NO:4) is used to link two zinc finger proteins.In another embodiment, the following linkers are used to link two zincfinger proteins: GGRR (SEQ ID NO:5) (Pomerantz et al. 1995, supra),(G4S)_(n) (SEQ ID NO:6)(Kim et al., Proc. Nat'l Acad. Sci. USA 93,1156-1160 (1996.); and GGRRGGGS (SEQ ID NO:7); LRQRDGERP (SEQ ID NO:8);LRQKDGGGSERP (SEQ ID NO:9); LRQKD(G₃S)₂ (SEQ ID NO:10). Alternatively,flexible linkers can be rationally designed using computer programcapable of modeling both DNA-binding sites and the peptides themselves(Desjarlais & Berg, Proc. Nat'l Acad. Sci. USA 90:2256-2260 (1993),Proc. Nat'l Acad. Sci. USA 91:11099-11103 (1994) or by phage displaymethods.

In other embodiments, a chemical linker is used to connect syntheticallyor recombinantly produced domain sequences. Such flexible linkers areknown to persons of skill in the art. For example, poly(ethylene glycol)linkers are available from Shearwater Polymers, Inc. Huntsville, Ala.These linkers optionally have amide linkages, sulfhydryl linkages, orheterofunctional linkages. In addition to covalent linkage of zincfinger proteins to regulatory domains, non-covalent methods can be usedto produce molecules with zinc finger proteins associated withregulatory domains.

In addition to regulatory domains, often the zinc finger protein isexpressed as a fusion protein such as maltose binding protein (“MBP”),glutathione S transferase (“GST”), hexahistidine, c-myc, and the FLAGepitope, for ease of purification, monitoring expression, or monitoringcellular and subcellular localization.

Expression Vectors and Introduction of Random Libraries into Cells

A. Cloning and Expression of Libraries Encoding Randomized Zinc FingerProteins

Nucleic acids encoding the randomized zinc finger proteins are typicallycloned into vectors for transformation into prokaryotic or eukaryoticcells for replication, expression, and cell transformation. Such vectorsare typically prokaryotic vectors, e.g., plasmids that act as shuttlevectors; eukaryotic vectors such as insect vectors, for storage,manipulation of the nucleic acid encoding zinc finger protein orproduction of protein; or eukaryotic vectors such as viral vectors(e.g., adenoviral vectors, retroviral vector, etc.) for expression ofzinc finger proteins and regulation of gene expression. The nucleic acidencoding a zinc finger protein can then be administered to a plant cell,animal cell, a mammalian cell or a human cell, a fungal cell, abacterial cell, or a protozoal cell.

To obtain expression of a cloned gene or nucleic acid, a zinc fingerprotein is typically subcloned into an expression vector that contains apromoter to direct transcription. Suitable bacterial and eukaryoticpromoters are well known in the art and described, e.g., in Sambrook etal., Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler,Gene Transfer and Expression: A Laboratory Manual (1990); and CurrentProtocols in Molecular Biology (Ausubel et al., eds., 1994). Bacterialexpression systems for expressing the zinc finger protein are availablein, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., Gene22:229-235 (1983)). Kits for such expression systems are commerciallyavailable. Eukaryotic expression systems for mammalian cells, plantcells, yeast, and insect cells are well known in the art and are alsocommercially available.

The promoter used to direct expression of a zinc finger protein nucleicacid depends on the particular application. Either a constitutive or aninducible promoter is used, depending on the particular use of the cloneencoding the zinc finger protein. Exemplary eukaryotic promoters includethe CaMV 35 S plant promoter, SV40 early promoter, SV40 late promoter,metallothionein promoter, murine mammary tumor virus promoter, Roussarcoma virus promoter, polyhedrin promoter, or other promoters showneffective for expression in eukaryotic cells.

The promoter typically can also include elements that are responsive totransactivation, e.g., hypoxia response elements, Gal4 responseelements, lac repressor response element, and small molecule controlsystems such as tet-regulated systems and the RU-486 system (see, e.g.,Gossen & Bujard, Proc. Nat'l Acad. Sci. USA 89:5547 (1992); Oligino etal., Gene Ther. 5:491-496 (1998); Wang et al., Gene Ther. 4:432-441(1997); Neering et al., Blood 88:1147-1155 (1996); and Rendahl et al.,Nat. Biotechnol. 16:757-761 (1998)).

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the nucleic acid inhost cells, either prokaryotic or eukaryotic. For example, regulatoryelements from eukaryotic viruses are often used in eukaryotic expressionvectors, e.g., SV40 vectors, papilloma virus vectors, and vectorsderived from Epstein-Barr virus.

A typical expression cassette thus contains a promoter operably linked,e.g., to the nucleic acid sequence encoding the zinc finger protein, andsignals required, e.g., for efficient polyadenylation of the transcript,transcriptional termination, ribosome binding sites, or translationtermination. Additional elements of the cassette may include, e.g.,enhancers, and heterologous spliced intronic signals.

The particular expression vector used to transport the geneticinformation into the cell is selected with regard to the intended use ofthe zinc finger protein, e.g., expression in plants, animals, bacteria,fungus, protozoa, etc. (see, e.g., viral expression vectors describedbelow and in the Example section). Standard bacterial expression vectorsinclude plasmids such as pBR322 based plasmids, pSKF, pET23D, andcommercially available fusion expression systems such as GST and LacZ. Apreferred fusion protein is the maltose binding protein, “MBP.” Suchfusion proteins are used for purification of the zinc finger protein.Epitope tags can also be added to recombinant proteins to provideconvenient methods of isolation, for monitoring expression, and formonitoring cellular and subcellular localization, e.g., c-myc or FLAG.

Some expression systems have markers for selection of stably transfectedcell lines such as neomycin, thymidine kinase, hygromycin Bphosphotransferase, and dihydrofolate reductase. High yield expressionsystems are also suitable, such as using a baculovirus vector in insectcells, with a zinc finger protein encoding sequence under the directionof the polyhedrin promoter or other strong baculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of recombinant sequences.

Standard transduction methods are used to produce bacterial, mammalian,yeast or insect cell lines that express the zinc finger proteins of theinvention. Transformation of eukaryotic and prokaryotic cells areperformed according to standard techniques (see, e.g., Morrison, J.Bact. 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983). These methods include thelipofection, microinjection, ballistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, agent-enhanced uptake of DNA, use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,plasmid vectors, viral vectors, both episomal and integrative, and anyof the other well known methods for introducing cloned genomic DNA,cDNA, synthetic DNA or other foreign genetic material into a host cell(see, e.g., Sambrook et al., supra, see also U.S. Pat. Nos. 5,049,386,4,946,787; 4,897,355; WO 91/17424, and WO 91/16024). It is onlynecessary that the particular genetic engineering procedure used becapable of successfully introducing at least one gene into the host cellcapable of expressing the protein of choice.

B. Viral Vectors

A preferred method of delivering the libraries of the invention to cellsis with viral vector delivery systems, including DNA and RNA viruses,which have either episomal or integrated genomes after delivery to thecell. The use of RNA or DNA viral based systems for the delivery ofnucleic acids encoding randomized zinc finger protein take advantage ofhighly evolved processes for targeting a virus to specific cells in thebody and trafficking the viral payload to the nucleus. Conventionalviral based systems for the delivery of zinc finger proteins couldinclude retroviral, lentiviral, adenoviral, adeno-associated, herpessimplex virus, and TMV-like viral vectors for gene transfer. Viralvectors are currently the most efficient and versatile method of genetransfer in target cells and tissues. Integration in the host genome ispossible with the retrovirus, lentivirus, and adeno-associated virusgene transfer methods, often resulting in long term expression of theinserted transgene. Additionally, high transduction efficiencies havebeen observed in many different cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system would thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6-10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),simian immuno-deficiency virus (SIV), human immuno-deficiency virus(HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In applications where transient expression of the zinc finger protein ispreferred, adenoviral based systems are typically used. Adenoviral basedvectors are capable of very high transduction efficiency in many celltypes and do not require cell division. With such vectors, high titerand levels of expression have been obtained. This vector can be producedin large quantities in a relatively simple system. Adeno-associatedvirus (“AAV”) vectors are also used to transduce cells with targetnucleic acids (see, e.g., West et al., Virology 160:38-47 (1987); U.S.Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy5:793-801(1994); Muzyczka, J. Clin. Invest. 94:1351(1994). Constructionof recombinant AAV vectors are described in a number of publications,including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol.5:3251-3260 (1985); Tratschin et al., Mol. Cell. Biol.4:2072-2081(1984); Hermonat & Muzyczka, Proc. Nat'l Acad. Sci. USA81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828(1989).

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and Ψ2 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by producer cell linethat packages a nucleic acid vector into a viral particle. The vectorstypically contain the minimal viral sequences required for packaging andsubsequent integration into a host, other viral sequences being replacedby an expression cassette for the protein to be expressed. The missingviral functions are supplied in trans by the packaging cell line. Forexample, AAV vectors typically only possess ITR sequences from the AAVgenome which are required for packaging and integration into the hostgenome. Viral DNA is packaged in a cell line, which contains a helperplasmid encoding the other AAV genes, namely rep and cap, but lackingITR sequences. The cell line is also infected with adenovirus as ahelper. The helper virus promotes replication of the AAV vector andexpression of AAV genes from the helper plasmid. The helper plasmid isnot packaged in significant amounts due to a lack of ITR sequences.Contamination with adenovirus can be reduced by, e.g., heat treatment towhich adenovirus is more sensitive than AAV.

In many situations, it is desirable that the vector be delivered with ahigh degree of specificity to a particular cell type. A viral vector istypically modified to have specificity for a given cell type byexpressing a ligand as a fusion protein with a viral coat protein on theviruses outer surface. The ligand is chosen to have affinity for areceptor known to be present on the cell type of interest. For example,Han et al., Proc. Nat'l Acad. Sci. USA 92:9747-9751 (1995), reportedthat Moloney murine leukemia virus can be modified to express humanheregulin fused to gp70, and the recombinant virus infects certain humanbreast cancer cells expressing human epidermal growth factor receptor.This principle can be extended to other pairs of virus expressing aligand fusion protein and target cell expressing a receptor. Forexample, filamentous phage can be engineered to display antibodyfragments (e.g., FAB or Fv) having specific binding affinity forvirtually any chosen cellular receptor. Although the above descriptionapplies primarily to viral vectors, the same principles can be appliedto nonviral vectors. Such vectors can be engineered to contain specificuptake sequences thought to favor uptake by specific target cells.

Assays for Determining Regulation of Gene Expression by Zinc FingerProteins

A variety of assays can be used to screen for phenotypic changes upontransduction of cells with the library encoding randomized zinc fingerproteins. A phenotype can be assessed by measuring, e.g., protein ormRNA levels, product levels, enzyme activity; transcriptional activationor repression of a reporter gene; second messenger levels (e.g., cGMP,cAMP, IP3, DAG, Ca²⁺); cytokine and hormone production levels using,e.g., immunoassays (e.g., ELISA and immunohistochemical assays withantibodies), hybridization assays (e.g., RNase protection, northerns, insitu hybridization, oligonucleotide array studies), colorimetric assays,amplification assays, enzyme activity assays, and other phenotypicassays.

For high throughput applications, typically either cells are pooled andtransduced in a batch, and then individually screened using flowcytometry, or the cells are pooled into clonal arrays and screened,e.g., with liquid robotics (see Example section). Examples of assays fora selected phenotype include e.g., changes in proliferation, anchoragedependence, growth factor dependence, foci formation, and growth in softagar; apoptosis assays, e.g., DNA laddering and cell death, expressionof genes involved in apoptosis; signal transduction assays, e.g.,changes in intracellular calcium, cAMP, cGMP, IP3, changes in hormoneand neurotransmitter release; receptor assays, e.g., estrogen receptorand cell growth; growth factor assays, e.g., EPO, hypoxia anderythrocyte colony forming units assays; enzyme production assays, e.g.,FAD-2 induced oil desaturation; pathogen resistance assays, e.g.,insect, bacterial, and viral resistance assays; chemical productionassays, e.g., penicillin production; transcription assays, e.g.,reporter gene assays; and protein production assays, e.g., VEGF ELISAs.

In one embodiment, the assay for the selected phenotype is performed invitro. In one preferred assay format, zinc finger protein regulation ofgene expression in cultured cells is examined by determining proteinproduction using an ELISA assay or an immunoassay such as fluorescenceactivated cell sorting.

In another embodiment, zinc finger protein regulation of gene expressionis determined by measuring the level of target gene mRNA expression. Thelevel of gene expression is measured using amplification, e.g., usingPCR, LCR, or hybridization assays, e.g., northern hybridization, RNaseprotection, dot blotting. RNase protection is used in one embodiment.The level of protein or mRNA is detected using directly or indirectlylabeled detection agents, e.g., fluorescently or radioactively labelednucleic acids, radioactively or enzymatically labeled antibodies, andthe like, as described herein.

Alternatively, a reporter gene system, e.g., that measures activation ofa gene in a pathway, can be devised using a promoter operably linked toa reporter gene such as luciferase, green fluorescent protein, CAT, orβ-gal. The reporter construct is typically co-transfected into acultured cell. After treatment with the zinc finger protein of choice,the amount of reporter gene transcription, translation, or activity ismeasured according to standard techniques known to those of skill in theart.

Identification and Isolation of Genes Associated with a SelectedPhenotype

After assaying for phenotypic changes, as described above, those cellsexhibiting an altered phenotype are selected for further study, in whichthe genes associated with the change in phenotype are identified andisolated. The genes are identified and isolated, e.g., usingdifferential gene expression analysis with microarrays; reversegenetics; e.g., identification of genes using zinc finger proteins toprobe YAC or BAC clones and using zinc finger proteins to scan genomicsequences; subtractive hybridization; differential cDNA cloningfrequencies, subtractive hybridization; by cloning ESTs from cells ofinterest; by identifying genes that are lethal upon knockout; byidentifying genes that are up- or down-regulated in response to aparticular developmental or cellular event or stimuli; by identifyinggenes that are up- or down-regulated in certain disease and pathogenicstates; by identifying mutations and RFLPs; by identifying genesassociated with regions of chromosomes known to be involved in inheriteddiseases; by identifying genes that are temporally regulated, e.g., in apathogenic organism; differences based on SNPs; by cross-linking thezinc finger protein to the DNA with which it is associated, followed byimmunoprecipitation of the zinc finger protein and sequencing of theDNA, etc.

In one embodiment, the candidate genes are identified by comparingpatterns of gene expression associated with the phenotypic change. Forinstance, down regulation of a gene by a ZFP-KRAB will result in underrepresentation of the corresponding mRNA when compared to a control(i.e., KRAB alone). There are several methods that can be employed tocompare patterns of gene expression including differential hybridizationscreening (see, e.g., Tedder et al., Proc. Nat'l Acad. Sci. USA85:208-212 (1988)), subtractive library construction (see, e.g., Daviset al., Nature 308:149-153 (1984)), representational difference analysis(RDA) (see, e.g., Hubank, Nucleic Acid Res 22:5640-5648 (1994));Lisitsyn et al., Science 259:640-648 (1993)) differential display (see,e.g., Liang et al., Nucleic Acid Res 21:3269-3275 (1993); Liang et al.,Science 257:967-971 (1992)), conventional cDNA array hybridization (see,e.g., Schummer et al., Biotechniques 23:1087-1092 (1997)) and serialanalysis of gene expression (SAGE) (see, e.g., Velculescu et al.,Science 276:1268-1272 (1997)).

In another embodiment, a technique called suppression subtractivehybridization (SSH) is used, which is a modification of the RDA as itnormalizes for mRNA abundance (see, e.g, Daitchenko et al., Proc. Natl.Acad. Sci. USA 93:6025-6030 (1996)). This technique will be used tocompare gene expression profiles of a target cell pre-and-post zincfinger protein transfection. This SSH cDNA library may be furtherscreened using microarrays containing oligonucleotide librariesrepresenting cDNA from relevant tissue types or, ultimately,oligonucleotides representing all open reading frames in the entiregenome. This combined screening of SSH cDNA libraries and microchiparrays screening will allow for the identification of putative functionsand pathway relationships for uncharacterized genes.

Bacterial artificial chromosomes (BAC) or yeast artificial chromosomes(YAC) containing large chromosomal segments representing the entirehuman genome can be employed to determine the gene (or genes)responsible for the observed phenotype. YAC or BAC clones containing thecandidate gene can be identified by physical capture using zinc fingerprotein or, alternatively, by probing arrayed clones.

Direct capture relies on physically binding and separating clonescontaining target DNA from the overall population of clones. Candidatezinc finger proteins are added to BAC or YAC libraries, using bufferconditions equivalent to those used in biochemical analysis of zincfinger proteins (see, e.g., U.S. Ser. No. 09/229,037, filed Jan. 12,1999, and Ser. No. 09/229,007, filed Jan. 12, 1999). Certain factorsshould be carefully adjusted so as to optimize specific binding by thezinc finger protein. Important chemical factors are zinc and salt(usually either potassium or sodium chloride). Zinc ion concentrationshould be 10 micromolar or less and salt should be 50 millimolar ormore. zinc finger protein and library DNA is added to the buffer. Theamounts of each reactant is important. Highest specificity is obtainedwhen the zinc finger protein is added at a concentration that is belowthe dissociation constant (as judged by gel shifts) of the protein forits designed target. The reaction is allowed to equilibrate at roomtemperature.

Modifications could include performing the initial binding at proteinconcentrations above the dissociation constant in order to maximizebinding. The process could be repeated using only the retained cloneswith concentrations of proteins that maximize specificity (i.e.,slightly below the dissociation constant). Another variation isseparating clones into pools rather than employing the entire library.The number of discrete clones in each pool would depend on the totallibrary size. For a library size of 1,000,000 clones, ten pools of100,000 clones or 100 pools of 10,000 clones and so on could beemployed. Following equilibration, the ZFP:DNA complex can be removedfrom the bulk solution by affinity capture of the zinc finger protein.Potential ligands are FLAG, MBP, biotin, 6×His (SEQ ID NO:11) or anyother tag for which an acceptable receptor exists. The receptor shouldbe immobilized to an inert support such as magnetic beads or sepharoseresin. Appropriate receptors would be FLAG antibody (FLAG epitope),amylose (MBP), streptavidin (biotin), nickel (6×His) (SEQ ID NO:11).

Once the clones are identified by capture they are sequenced to identifycoding regions. The genomic inserts cloned into the BACs or YACs may betoo large to pinpoint the exact gene responsible for the phenotype. Thelist of possible candidate genes within a clone could be narrowed in thecases where clones with overlapping, but not identical, sequences werecaptured. Only the regions common to both clones should contain thecandidate genes. Alternatively, clones containing smaller segments ofeach BAC and YAC could then be used for capture. Other vectors usedcould include lamba, P1 or cosmids.

In another embodiment, physical capture and retention of DNA in solutionis an array-based method where zinc finger proteins are used as probesto detect clones possessing the correct target sequences. BAC and YAClibraries would be arrayed so that each clone would occupy an uniqueaddress on a support such as glass, nitrocellulose or any other materialwhich allows nondestructive immobilization of DNA. The zinc fingerproteins would be conjugated to a fluorophore either pre- orposttranslationally. The supports containing the clones would be floodedwith the zinc finger protein and incubated for a sufficient time toallow binding. Then unbound zinc finger protein would be washed offusing conditions that minimize non-specific binding. Binding would bevisualized by exposing the filter to an appropriate wavelength of light,exciting the fluorophore to emit at a characteristic wavelength.

This method could be refined by simultaneously adding two zinc fingerproteins labeled with different fluorophores. By using the fluorophoresemitting at appropriate wavelengths, binding of both zinc fingerproteins to the same clone could be detected simultaneously bymonitoring the output color which should be a combination of bothwavelengths. For instance, the presence of a blue emitting fluorophoreand a yellow emitting fluorophore would produce green light. Flurophorescould be fluorescent proteins that are modified from green fluorescentprotein to produce the spectrum of wavelengths. Alternatives would befluorescent dyes with reactive groups that can be conjugated to proteinmoieties post purification or fluorescently labeled antibodies. As withphysical capture, once a BAC or YAC clone is identified differentregions can be probed in sublibraries with shorter inserts.

In another embodiment, physical capture is achieved by tagging DNAtargets which are bound by their specific zinc finger protein by using amodified catalyzed reporter deposition (CARD) method. CARD has been usedas a means of signal amplification in immunocytochemistry, ELISA, andblotting (Adams, J. Histochem. Cytochem. 40:1457-1463 (1992); Bobrow etal., J. Immunol. Meth. 150:145-149 (1992)). This technique normallyinvolves the use of horseradish peroxidase (HRP) in the presence ofhydrogen peroxide to catalyze biotinylated tyramine deposition aroundthe site of the enzyme activity. This results in biotinylation ofmolecules or motifs that are proximal to the active enzyme. Thistechnique has been adapted to allow the specific recovery of neighboringphage antibodies binding around a core ligand binding site on a cellsurface (Osbourn et al., Nat. Biotech. 16:778-781 (1998)).

A modified CARD technique would be used to biotinylate genes which havebeen recognized by the randomized zinc finger proteins of the invention.The zinc finger proteins could be either directly engineered as HRPfusions, or HRP conjugated antibodies which recognize the zinc fingerproteins (by their FLAG sequence for instance) could be used. HRPconjugated anti-FLAG monoclonal antibody and biotin tyramine are addedto an equilibrated solution of zinc finger proteins and libraries. Ineither scenario, the biotin which is covalently attached to DNAsequences surrounding the zinc finger proteins, and this biotin “tag”will provide a handle for further manipulation. The biotinylated DNAscan be captured and purified with streptavidin-coated magnetic beads(Dynal, Oslo). Another way to capture the DNAs that are recognized byzinc finger proteins is to use an anti-FLAG affinity column to purifythe DNAs. Post capture the DNAs will be cloned, sequenced and otherwisecharacterized.

In another embodiment, genes are identified by scanning genomicsequences. The target gene sequence can be predicted based on therecognition residues of each zinc finger. By using these rules for aminoacid side-chain contacts with nucleotide bases, the nucleotide sequencecan be “read off” of the zinc finger protein. Allowances for ambiguitiescan be made based on a knowledge of specificity for each interaction orcombinations of interactions. Genes can be identified by searching theGenbank DNA database (National Center of Biotechnology Information) formatching sequences using an algorithm such as BLAST (Altschul et al., J.Mol. Biol. 215:403-410 (1990)). Ultimately it will be possible to searchthe whole human genome. The expectation is that many of the zinc fingerproteins targeting candidate genes will recognize different sequences ofthe same gene or genes. Thus, confirmation that any one zinc fingerprotein is truly targeting a particular gene is obtained by grouping thegenes identified by different zinc finger proteins and deriving aconsensus.

In another embodiment, genes are identified by cross linking the zincfinger protein and the nucleic acid (or chromatin) to which it is bound,immunoprecipitating the cross-linked complex, and then sequencing thenucleic acid to identify the gene of interest. Sequence-specific bindingof a zinc finger protein to chromatin is assayed, e.g., by chromatinimmunoprecipitation (ChIP). Briefly, this technique involves the use ofa specific antibody to immunoprecipitate chromatin complexes comprisingthe corresponding zinc finger protein antigen, and examination of thenucleotide sequences present in the immunoprecipitate.Immunoprecipitation of a particular sequence by the antibody isindicative of interaction of the zinc finger protein antigen with thatsequence (see, e.g., O'Neill et al. in Methods in Enzymology, Vol. 274,pp. 189-197 (1999); Kuo et al., Method 19:425-433 (1999); and Ausubel etal., supra, Chapter 21).

In one embodiment, the chromatin immunoprecipitation technique isapplied as follows. The zinc finger protein is introduced into a celland, after a period of time sufficient for binding of the zinc fingerprotein to its binding site has elapsed, cells are treated with an agentthat crosslinks the zinc finger protein to chromatin if that molecule isstably bound. The zinc finger protein can be crosslinked to chromatinby, for example, formaldehyde treatment or ultraviolet irradiation.Subsequent to crosslinking, cellular nucleic acid is isolated, shearedand incubated in the presence of an antibody directed against the zincfinger protein. Antibody-antigen complexes are precipitated, crosslinksare reversed (for example, formaldehyde-induced DNA-protein crosslinkscan be reversed by heating) and the sequence content of theimmunoprecipitated DNA is tested for the presence of a specificsequence, for example, the target site of the zinc finger protein.

In a preferred embodiment, the immunoprecipitated DNA is tested for thepresence of specific sequences by a sensitive hydrolyzable probe assayallowing real-time detection of an amplification product, knowncolloquially as the Taqman® assay. See U.S. Pat. No. 5,210,015; Livak etal., PCR Meth. App. 4:357-362 (1995); and Heid et al., Genome Res.6:986-994 (1995). Briefly, an amplification reaction (e.g., PCR) isconducted using a probe designed to hybridize to a target sequenceflanked by two amplification primers. The probe is labeled with afluorophore and a fluorescence quencher such that, when not hybridizedto its target sequence, the probe does not emit detectable fluorescence.Upon hybridization of the probe to its target and hydrolysis of theprobe by the polymerase used for amplification, the fluorophore isreleased from the vicinity of the quencher, and fluorescence increasesin proportion to the concentration of amplification product. In thisassay, the presence of increased levels of an amplification productcorresponding to the binding site for the zinc finger protein, comparedto levels of amplification product specific to a control genomicsequence, is indicative of binding of the zinc finger protein to itsbinding site in cellular chromatin.

Additional methods for detecting binding of zinc finger protein tochromatin include, but are not limited to, microscopy (e.g., scanningprobe microscopy), fluorescence in situ hybridization (FISH) and fusionof a DNA methylase domain to the zinc finger protein, in which casesequences to which the zinc finger protein is bound become methylatedand can be identified, for example, by comparing their sensitivity tomethylation-sensitive and methylation-dependent restriction enzymes orby using antibodies to methylated DNA. See, for example, van Steensel etal., supra.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of noncritical parameters that could be changed or modified toyield essentially similar results.

Example 1 Protocol for Preparation and Screening Using a Randomized ZincFinger Protein Library Generated by Finger Grating

A. Generation of a Library Using Finger Grafting

A library of 12 different helices compatible with 5 different fingerpositions will be created and assembled into zinc finger proteins usinga method similar to that currently used to assemble engineered 3 fingerproteins (see, e.g., U.S. Ser. No. 09/229,037 filed Jan. 12, 1999, andU.S. Ser. No. 09/229,007, filed Jan. 12, 1999). Randomness will beconfirmed by sequencing a representative sample. Of this library,250,000 individual bacterial transformants will be picked and archived.The individual transformants will be combined into pools of 8 and clonedinto a viral delivery vector (such as an adenoviral vector).

Viral delivery particles will be produced from each pool (there are31,250 different pools) and tested in an appropriate assay for theidentification of a desired phenotype. Assays could be the developmentof growth factor independence, secretion of EPO, angiogenesis, apoptosisetc.

Biologically active zinc finger proteins (“hits”) will be confirmed bysecondary screening. The gene directly responsible for the phenotypewill be identified either by virtue of proximity of a binding site (thebinding site for the active zinc finger protein can be surmised by helixcomposition or determined experimentally by site selection) if thesequence is known, or pulled from a genomic library using the zincfinger protein itself as a molecular probe.

Specific recognition helices for twelve different DNA triplet sequenceshave been characterized. These helices are referred to by their “SBS”numbers. The table below shows the target DNA triplet sequences and theamino acid composition of the twelve different recognition helices. Any5-finger zinc finger protein comprising a unique subset of 5 of these 12recognition helices will recognize a distinct and unique 15 basepair DNAsequence.

TABLE 1 SBS Number Target Triplet Recognition Helix SEQ ID NO: SBS1 GTGRSDALTR 12 SBS2 GAG RSDNLAR 13 SBS3 GGG RSDHLSR 14 SBS4 GCG RSDELTR 15SBS5 GCA QSGSLTR 16 SBS6 GCT QSSDLTR 17 SBS7 GCC ERGTLAR 18 SBS8 GATQSSNLAR 19 SBS9 GAC DRSNLTR 20 SBS10 GAA QSGNLAR 21 SBS11 GGC DRSHLAR 22SBS12 GGA QSGHLQR 23

For example, a zinc finger protein made up of (reading from theC-terminus) SBS 4-10-9-10-2 would recognize the DNA sequence (reading 5′to 3′) GCG GAA GAC GAA GAG (SEQ ID NO:24).

DNA encoding each of the 12 SBS helices are synthesized asoligonucleotides. These oligonucleotides are mixed in equimolar amountsand combined with oligonucleotides that encode the remaining amino acidsof a five finger zinc finger protein based on the amino acid sequence ofthe murine zinc finger protein Zif268. This mixture is PCR amplified asdescribed in U.S. Ser. No. 09/229,037, filed Jan. 12, 1999, and U.S.Ser. No. 09/229,007, filed Jan. 12, 1999, and subcloned into twodifferent mammalian expression such that one vector produces a chimerictranscription factor comprising a nuclear localization sequence, thezinc finger protein DNA binding domain, the VP16 activating domain andthe FLAG epitope tag (this vector is referred to using an acronym of itscomponent parts; NVF). The second vector is identical to the firstexcept that a KRAB transcriptional repression domain replaces the VP16domain (NKF). The rest of the vector sequences support the production ofvirus-based delivery components such as the sequences required forrecombination into adenoviral vectors and packaging into viral particles

These vectors are used to transform E. coli. Individual colonies,representing distinct individual zinc finger protein clones, are pickedand subcultured in 96-well microtiter dishes. 250,000 clones are pickedand arrayed for each vector system (NVF and NKF). This creates anarrayed zinc finger protein library comprising approximately 2,600microtiter plates. These libraries are stored as glycerol stocks at −80°C.

DNA sequence analysis of a subset of each library confirms that the fivefinger zinc finger proteins encoded represent a random assortment of the12 recognition helices.

The zinc finger protein E. coli clone library is converted to a pooledviral delivery library as follows. The E. coli clones are arranged intopools of 8 different clones by pipetting adjacent wells together using a12-channel multi-channel pipette (this can be done robotically). Thepools are grown in rich medium using deep-well microtiter dishes at 37°C. Plasmid DNA is prepared using Qiagen columns. The DNA pools are thenused to transfect PERC.6 cells (a cell line used to produce adenoviralvectors). Several days later the viral vector-laden culture supernatantsare collected and stored at −80° C.

B. Screening for the Selected Phenotype

An assay for a particular desired phenotype is now created andimplemented using a microtiter-based method. The viability of a growthfactor dependent cell line, capable of detecting autocrine production ofa growth factor such as EPO or VEGF is one such assay, described below.

Once the assay is created, the influence of the zinc finger proteinlibrary members on the assay can be determined using robotic methodscommonly employed in the high throughput screening industry. A sample,in this case a pool of 8 different zinc finger proteins carried inadenoviral delivery vectors, is added to a well of an assay plate, inthis case a growth factor dependent cell line in minimal medium. Severaldays later the assay plate is tested to determine if any of the zincfinger proteins caused the cell line to grow. Growth can be determinedusing many different high throughput assays, in this case by themetabolic conversion of a fluorescent dye Alamar Blue.

Hits from the high throughput assay (wells where cell growth wassupported) are confirmed by simply retesting the pool and then the poolis “deconvoluted,” separating it into individual zinc finger proteincomponent members and retested to determine which of the 8 zinc fingerproteins triggered cell growth.

Once a zinc finger protein-phenotype connection has been established,mechanistic and genomic analyses can be performed to identify the generesponsible for the phenotype. In this case, the independence of growthfactors suggests autocrine production of a growth factor. This can besimply confirmed by testing the growth supporting nature of zinc fingerprotein treated conditioned medium on otherwise untreated growth factordependent cells.

After the autocrine mechanism has been confirmed, the task becomes oneof determining which growth factor gene was switched on by the zincfinger protein library member. Well characterized growth factors can beeliminated by using inactivating antibodies. Suspect genes can beidentified by scanning the sequence databases for the 15 basepairsrecognized by the active zinc finger protein. This sequence can bedetermined either by simply reading off the recognition helices' aminoacid sequence and predicting the DNA target sequence using therelationships outlined in the table above, or by using site selectionexperiments as described in previous applications to determine the DNAtarget sequence empirically. Suspect (or candidate) genes can also beidentified using experimental method designed to measure globaldifferential gene expression (such as gene expression microarrays).Finally, the zinc finger protein itself can be used as a probe for YACor BAC clones to identify candidate loci.

C. Screening Using Flow Cytometry

In addition to screening using microtiter type assays (as describedabove), flow cytometry and cell sorting can be used to screen forspecific phenotypes. A flow cytometer simply measures the fluorescenceof one cell at a time as a stream of cells flow past a laser. Multiplelasers and multiple detectors permit simultaneous detection of severalfluorophores (typically up to 4). A wide variety of fluorescent probeshave been developed allowing the measurement of cell surface markers,DNA content, green fluorescent protein and other cytoplasmic components.Multi-marker analysis allows one to study a specific cell population,defined by specific cell surface markers, in complex mixtures of cellssuch as whole blood. In addition to simply detecting the kind andintensity of specific fluorescent markers on cells as they flow past thelaser beams, cytometers with sorting capability can collect specificpopulations of cells one cell at a time. This permits the outgrowth ofvery specific cell populations (if the labeling method is not toxic, notalways the case) and/or the application of bulk-type assays (westernblots, northern blots etc.) on homogeneous and very specific populationsof cells.

In screening, a cell sorter permits the isolation of a single cell or apopulation of cells displaying a desired phenotype. This could be theappearance of a specific receptor on the surface of a cell treated by aspecific cytokine (i.e. the appearance of ICAM on the surface of cellstreated with IL-1) or any other measurable response.

In practice, a library is created in retroviral vectors. This could bethe same zinc finger protein library described above. Susceptible cells(for example U937 monocytic cells) are transduced using theretroviruses. A specific phenotype is detected using flow cytometry andcells displaying the desired phenotype collected into separate wells ofa microtiter plate. The zinc finger proteins causing the desiredphenotype can then identified by rescuing the retroviral sequences usingPCR.

Example 2 Protocol for Preparation and Screening Using a Randomized ZincFinger Protein Library Generated by Codon Doping

A. Preparation of the Library Using Codon Doping

As described above, each zinc finger binds three nucleotides using fourcritical amino acids in the recognition helix. If each base in thecodons for these amino acids was simply randomized, it would generate alibrary of 4¹² clones (1.7 million). This number is already in excess ofa desired library limit of about one million to about 10 or 100 millionclones and only concerns one finger (and three are to be used in thesemethods). However, it is not necessary to use completely random codons.Because of the redundancy of the genetic code, schemes ofsemi-randomization can generate representatives of all, or nearly allcodons. This strategy is thus called a codon doping scheme.

One randomization scheme uses VNS instead of NNN, where N=any base, V=Aor G and S=G or C. All of the codons are represented by VNS except Phe,Trp, Tyr, Cys and all translation termination codons. It is advantageousto eliminate the termination codons and loss of the four amino acidslisted is tolerable because they are typically underrepresented in knownprotein DNA contacts. With the VNS scheme it is possible to randomize 4amino acids in significantly less than a million clones (331,776 to beexact). However, varying a fifth position pushes the library size intothe 8 million clone range. Some finger positions will still need to befixed. The four critical amino acids of finger 1 will be randomizedusing the VNS scheme and fingers 2 and 3 will be fixed to recognize theDNA sequence GGG GAG. Specific fingers for these triplets are availablethat do not recognize alternative binding sites. This 6 base pairanchoring sequence will occur once every 4⁶ (4096) bases and should liewithin a reasonable distance of the transcription initiation site ofmost genes. The randomized finger will direct the zinc finger proteinsto subsets of these anchoring sites with 3 or 4 additional bases ofsequence specificity. In future experiments additional libraries can beexamined that carry alternative anchoring fingers.

The mutagenesis strategies proposed to generate the three-finger zincfinger protein library is represented below:

TABLE 2 −1 1 2 3 4 5 6 Finger 1 VNS S VNS VNS L A VNS Finger 2 R S D N LA R (SEQ ID NO: 13) Finger 3 R S D H L S R (SEQ ID NO: 14)

To balance the diversity and size of the zinc finger protein library,the relatively highly conserved serine is fixed at position 1; leucineat position 4 (which does not contact DNA but is involved in stabilizingthe fold of the finger); and a small alanine at position 5. All therandomization will be built in by polymerase chain reaction using 3degenerate oligos (2, 4, and 6) that contain the VNS dope schemes forthe −1, 2, 3, and 6 positions (FIG. 1).

Codon Doping Protocol

1. Dilute the following oligos to 0.5 μM in H₂O

Oligo 1: SCOM (Sangamo Common Oligo) 1

Oligo 2: Oligo encoding randomized finger 1: (VNS)S(VNS)(VNS)LA(VNS),see text for explanation of notation.

Oligo 3: SCOM 2

Oligo 4: Oligo encoding finger 2: RSDNLAR (SEQ ID NO:13)

Oligo 5: SCOM 3

Oligo 6: Oligo encoding finger 3: RSDHLSR (SEQ ID NO:14)

2. Set up PCR reactions as follows:

50 μl 2×PCR Master Mix, Boehringer Mannheim

-   -   1 μl SCOM 1    -   1 μl SCOM 2    -   1 μl SCOM 3    -   1 μl Randomized Finger 1 oligo    -   1 μl Finger 2 oligo    -   1 μl Finger 3 oligo

44 μl H₂O

100 μl total volume

3. Run the following PCR program to form the initial “scaffold” ofoligos (see diagram):

95° C. 5 minutes; 95° C. 30 seconds; 40° C. 30 seconds×4 cycles

72° C. 1 minute

4. Then add external primers (SCOM F, at 10 μM, and SCOM R, at 10 μM), 2μl of each primer (refer to diagram).

5. Continue with the following PCR program:

95° C. 1 minute; 95° C. 30 second; 62° C. 30 seconds×30 cycles

72° C. 1 minute

72° C. 10 minutes

4° C. soak

6. Run entire reaction through Qiagen PCR Clean-up column. Elute in 50μl H₂O

7. Set up Kpn I/Bam HI restriction digest:

50 μl clean PCR product

10 μl NEB Bam HI Buffer, 10×

10 μl NEB BSA, 10×

3 μl NEB Kpn I restriction enzyme, u/l

2 μl NEB Bam HI restriction enzyme, u/l

25 μl H₂O

100 μl total volume, incubate at 37° C. for 4 hours.

8. Run entire digest on a 1.4% agarose gel (split sample into twolanes). Gel extract and purify the 300 bp fragment from each lane usingQiagen Gel Extraction Kit. Elute each in 30 μl H₂O, then combine fortotal volume of 60 μl.

9. Ligate into a phage vector such as SurfZAP (Stratagene) that has beenmodified to possess Kpn I and Bam HI restriction sites in theappropriate frame as to generate a plasmid encoding ZFP-cpIII fusionprotein.

10. Transform into XL-1 Blue bacteria and plate onto LB+100 g/mlampicillin. Grow overnight at 37° C.

11. Pick individual colonies and sequence to ensure that finger 1randomization is sufficiently represented.

B. Packaging into Viral Vectors for Delivery

This step entails cloning the zinc finger protein libraries from thedonor phage vectors into an AAV (adeno-associated viral) vector. Eachvector will retain an intact cis-acting ITR sequence, followed by acytomegalovirus promoter. The ITR sequences are required in cis toprovide functional origins of replication (ori) as well as the signalsfor encapsidation, integration into the cell genome and rescue fromeither host cell chromosomes or recombinant plasmids. To maintain anoptimal wild-type AAV genome size for the vectors, an additional,functionally inert intron sequence will be incorporated into the DNAconstruct. This intron will be spliced out in the final mRNA that wouldencode the functional zinc finger protein. The zinc-finger genes will bemodified to incorporate a Kozak sequence for proper translationinitiation and add a nuclear localization sequence such as that from theSV40 T antigen. The sequence for the assembled zinc finger proteinexpression constructs will be as follows: Kozaksequence-NLS-ZFPs-KRAB/VP16-FLAG.

Two distinguishable phases of the AAV life cycle can occur in permissiveor non-permissive conditions (see FIG. 3). In permissive cells, thepresence of a helper virus, typically adenovirus, causes an infectingAAV genome to be greatly amplified generating a large burst ofinfectious progeny. This biological property will be exploited togenerate AAV-ZFP vectors at genomic scale as well as to rescue insertsfrom relevant target cells if needed.

In a productive infection, the infecting parental AAV single strandgenome is converted to a parental duplex replicating form (RF) by aself-priming mechanism which takes advantage of the ability of the ITRto form a hairpin structure. This process can occur in the absence ofhelper virus but is enhanced by a helper virus. The parental RF moleculeis then amplified to form a large pool of progeny RF molecules in aprocess which requires both the helper functions and the AAV rep geneproducts, Rep78 and Rep68. AAV RF genomes are precursors to progenysingle strand (SS) DNA genomes that are packaged into pre-formed emptyAAV capsids composed of VP1, VP2 and VP3 proteins.

In the absence of a helper virus the AAV genomes reach the cell nucleusbut bulk replication generally does not occur. The infecting genomes areconverted to double stranded DNA (dsDNA) and may persist as freeunintegrated genomes for a considerable number of cell passages.Expression of exogenous vector genes can occur from these dsDNA formsand these vector sequences can be rescued through packaging into newviral particles. These new viral particles are generated by induction ofcell permissiveness through infection with a helper viruses ortransfection with plasmids which express all of the appropriate helperfunctions. This biological characteristic allows rAAV particle recoveryby amplification from a target cell. Therefore, subsequent isolation andcharacterization of viruses expressing desired sequences is accomplishedin a rapid and facile manner.

Protocol for Generating rAAV-ZFP Library

1. Isolate “library” of zinc finger protein inserts from zinc fingerprotein phage library DNA prep by digesting with Kpn I and Bam HIrestriction enzymes.

2. Ligate the Kpn I/Bam HI ZFP-encoding fragment into the abovementioned AAV vectors. Each AAV vector has already been modified topossess the NLS-Kpn I site-Bam HI site-VP16 or KRAB-FLAG. Thereby, theresulting ligations should result in plasmids encoding NLS-ZFPs-KRAB orVP16-FLAG.

3. Starting with the repressor library (KRAB), transform theAAV-ZFP-KRAB plasmids into XL-1 Blue bacteria. Grow overnight on plates.

4. Pick resulting colonies and array into 96-well format for small-scalebacterial cultures (refer to step 1, FIG. 4).

5. Isolate AAV-ZFP-KRAB plasmids, maintaining the 96-well arrayedformat.

6. Plate 293 cells, already stably expressing the AAV rep and cap geneproducts, in 96-well format. This should be done the day prior totransfection.

7. Infect 293 cells with Adenovirus (Ad), incubate for 1 hour at 37° C.

8. Using the DEAE-Dextran transfection technique, cotransfect theAAV-ZFP-KRAB plasmids along with helper plasmid encoding for the AAV repand cap gene products. Add the DNA-DEAE-Dextran solution directly to theinfected cells. Incubate for 4-5 hours at 37° C. Wash cells andreplenish with complete media. Incubate for 72 hours.

9. To recover the resulting AAV-ZFP-KRAB viruses (rAAV-ZFP-KRAB),harvest and lyse the cells (see, e.g., Matsushita et al., Gene Therapy5:938-945 (1998)). Clear the lysate of cellular debris by a low speedcentrifugation spin and heat inactivate the Ad virus. The arrays ofrAAV-ZFP libraries can be stored at −70° C. until assayed.

C. Selecting a Phenotype of Interest

The cells transfected with rAAV-ZFPs will each be expressing differentgenes in different levels, resulting in different phenotypes. Generally,one is interested in a specific phenotype that can be identified easilyin a high throughput (HTP) assay. Numerous assays have been developedwhich can identify changes in cell growth and metabolism. The assayemployed depends on the pathway of interest. Once a cell expressing thedesired phenotype is identified, the genes expressed/repressed can bedetermined.

Assay for Target Discovery Using Inhibition of VEGF Induction DuringHypoxia

Vascular Endothelial Growth Factor (VEGF) is the principlepro-angiogenic factor responsible for eliciting the growth of new bloodsupply to hypoxic tissues. VEGF expression is triggered by hypoxia in awide variety of cell types. This regulation occurs principally at thelevel of transcription. The hypoxic triggering of VEGF gene expressionis central to several important pathologies both in a negative andpositive sense. Blockade of VEGF induction could lead to the treatmentof solid tumor growth and diabetic retinopathy. Thus, in this example,factors that inhibit hypoxic stimulation of VEGF are identified.

The human embryonic kidney epithelium-derived cell line 293 can beinduced to secrete VEGF into the growth medium by making the cellshypoxic or by mimicking hypoxia using cobalt chloride. This inductioncan be followed using a simple ELISA.

293 cells, previously stably transfected with a gene expressing secretedalkaline phosphatase (SEAP), will be plated in a 96-well format. Thecultures will be transduced with the rAAV-ZFP-KRAB library, alreadyarrayed in 96-well format (see above), and allowed to incubate for 48hours. Next, VEGF expression will be induced using CoCl₂. 24 hours postVEGF-induction, culture supernatants will be tested for VEGF secretion.In addition, the secretion of SEAP will be examined as a general controlfor toxicity and secretion function. Cells that fail to induce VEGFexpression will be scored as primary hits.

The zinc finger proteins responsible for the primary hits will berecovered and retested in secondary assays confirming the specificblockade of the VEGF inducing hypoxic signal (Target Validation). Inthis case, a HTP ELISA is employed to identify the desired phenotypicresponse in the presence of a specific AAV-ZFP that has targeted a geneinvolved in hypoxic stimulation of VEGF.

Assay for Target Discovery Using Up-Regulation of E-Cadherin on the CellSurface

E-cadherin is a focal point in the development of numerous cancers andits function is frequently inactivated in the development of breast,colon, prostate, stomach, liver, esophagus, skin, kidney and lungcancers amongst others. The loss of E-cadherin function is a ratelimiting step in the transition of cells from well differentiatedadenoma to invasive carcinoma cells. Chromatin rearrangement, mutation,hypermethylation, and loss of transcription-factor binding are allthought to play roles in suppression of E-cadherin function.Furthermore, alterations in function, expression levels, and signalingproperties of molecules which associate with E-cadherin have also beenshown to play a role in this loss of function. This widespread loss offunction in numerous cancer types implies a profound role for E-cadherinin these cancers where it is manifested by de-differentiation, increasedinfiltrative growth and metastatic potential. Re-establishment ofE-cadherin function in various cell culture and in vivo systems hasdemonstrated the reversion of invasive tumors to a benign, epithelialphenotype. Therefore, in this example, genes which could be invoked toup-regulate E-cadherin expression are identified.

The cell line which will be selected for use in the phenotypic screeningassay must be able to express E-cadherin at its surface upon inductionof expression by the specifically constructed zinc finger motif. In thiscase, the HT-29 human colon carcinoma cell line, which has been shown toupregulate E-cadherin expression in response to dimethylsulfoxide (DMSO)in a dose dependent manner, would be appropriate.

Once again, cells are plated in a 96-well format. This time, the cellsare transduced with members of a rAA-ZFP-VP16 library, produced asdescribed above. They will be examined for the presence of cell-surfaceexpression of E-cadherin 48 hours post-transduction. Treatment of thecells with DMSO would serve as a positive control.

Determining cell-surface E-cadherin expression can be done by one ofseveral methods. One method is accomplished by binding fluorescentlytagged antibodies directed against the E-cadherin on the cell surface.Quantitation of this fluorescence is then determined by a 96-wellfluorometer. Alternatively, a relatively less sensitiveimmunohistochemical assay performed in a 96 well format may besufficient for evaluation of up-regulation, supporting the premise ofthis approach. Another approach to assaying the upregulation ofE-cadherin is based on proteolytic digestion of a fluorescence labeledprotein substrate. This assay has the potential of being simpler andmore sensitive than the one based on using antibodies to detectE-Cadherin expression. It has been shown that in some cancer cell typessecreted matrix metalloproteinases are down regulated by theupregulation or reconstitution of E-cadherin expression. In the proposedhigh throughput assay system, a fluorescently tagged protein substrate(Molecular Probes, EnzChek Assay Kit) does not fluoresce because of thequenching phenomena observed when numerous fluorescent tags are in closeproximity to one another. However, when this labeled protein substrateis cleaved by proteases, a fluorescent signal is observed whichcorresponds to the proteolytic activity in the sample. For screeningpurposes positive hits would be counted where fluorescence emission isquenched indicating down regulation of protease activity. These positivesamples would then be further analyzed and tested for E-cadherinexpression.

The up-regulation of E-cadherin would represent the generation of thedesired change in tumor cell phenotype induced by the zinc fingerprotein's action on a gene(s) expression. Thus, indicating that thisgene(s) may prove to be a good candidate for drug discovery.

D. Identifying Candidate Genes Associated with a Selected Phenotype

Once a “hit” has been identified, using, e.g., one of the assaysdescribed above, one must then determine the gene(s) the zinc fingerprotein has influenced that resulted in the desired phenotype. The firststep is to identify the zinc finger protein that was involved. This iseasily accomplished as indicated in the previous section referring torAAV recovery. By infecting the cells containing the AAV-ZFP of interestwith helper virus, the AAV will enter a lytic cycle and thereby produceprogeny virus. Isolation of these rAAV particles from the target cellcan be done as previously described. This assures that there is plentyof the rAAV-ZFP for additional experiments and manipulations. Analysisof the zinc finger protein can suggest a putative recognition targetsite that when compared to sequences listed in GenBank could identifygenes that may be affected by the zinc finger protein.

Comparing mRNA of ZFP-transduced vs. non-transduced cells is a directway of identifying differentially expressed genes. Several methods havebeen developed to do this sort of analysis: subtractive hybridization,differential display and array analysis, as described above.

1. A nucleic acid library encoding randomized zinc finger proteins, eachzinc finger protein comprising between 3 and 5 zinc fingers, and whereineach zinc finger comprises a non-naturally occurring recognition helixsequence of known binding specificity for a 3 base pair target nucleicacid site.
 2. The library of claim 1, wherein each member of the libraryfurther encodes a functional domain.
 3. The library of claim 2, whereinthe functional domain is an activation domain.
 4. The library of claim2, wherein the functional domain is a repression domain.
 5. A eukaryoticcell population transfected with the library of claim
 1. 6. The cellpopulation of claim 5, wherein each member of the library furtherencodes a functional domain.
 7. The cell population of claim 6, whereinthe functional domain is an activation domain.
 8. The cell population ofclaim 7, wherein the functional domain is a repression domain.
 9. Thelibrary of claim 1, wherein each zinc finger protein contains three zincfingers.